Plant Tissue Culture and the Effects of Auxin Hormone on It - Term Paper Example

Plant tissue culture and the effects of Auxin hormone on it Abstract Tissue culture proves to be the most challenging aspect in transformational strategies in plants. However, it can not be assumed as it is an integral part of transformational strategies in plants. Development of efficient regeneration strategies is very necessary in order to avoid numerous deleterious effects caused by somaclonal variation. Regeneration protocols vary with transformational techniques. There are crops which are only amenable to only one specific protocols while others are amenable to many transformational and regeneration strategies. The technology can not be ignored as its benefits outweigh the cost if a cost-benefit analysis is done. Plant tissue culture has played a great role in reducing food insecurity, providing jobs and an overall advantage on the environment. In the countries where this is practiced, the country achieves maximum financial benefits. List of contents Abstract . List of Tables Error: Reference source not found List of Figures Error: Reference source not found 1.4 Particles of the Tissue Culture Medium 9 1.5 Preparation of Media 11 Source: Slater et al. (2008) 20 20 3.Culture types 20 3.1 Callus Induction 20 3.3The importance of plant tissue culture 22 Appendix Error: Reference source not found List of Tables Page Table (2):media for plant tissue culture 7 List of Figures Page Figure(1): IAA 10 Figure(2): (2) (NAA) , (2,4-D) , 4-CI-IAA, PAA 10 Fig.(3):Microscopic image of the Apical meristem 11 Fig.(4):show Coleoptile 12 Fig.(5):Important experiments of auxin in plant 15 Fig.(6) :The effect of different ratios of auxin to cytokinin on the growth and morphogenesis of callus. 15 Introduction The growth and development of plants is regulated by a mixture of genetic factors and environment influences like all organisms. However, some plants have long life and ability to adapt to the environmental conditions than have animals. Over the last few decades, the technology of plant tissue culture has become very common in many countries because by this technique it is possible to produce extra copies of a plant and production plants without the use of seeds. The first successful plant tissue culture was at the end of 20th century by Haberlandt, when he reported the culture of leaf mesophyll tissue and hair cells. Moreover, one of the most significant factors is auxins (IAA) that are the first plant growth regulators. It is necessary in formation of roots and shoot, in phototropism, fruit development, abscission, initiation and development of roots, gravitism, embryo development and providing a shade avoidance effect. Later, there were several reported successful cases of plant tissue culture but the progress was disrupted by World War two. The purpose of this project, therefore, is to examine the effects of Auxin hormone on the plant tissue culture in terms of the differences in percentage of auxin to cytokinin on the growth of callus. In this project, there are three sections; the first section will discuss the definition of plant tissue culture, concepts that are central to understanding plant cell culture, some of elements that are essential Prepare the tissue culture medium. The second section deals with the aspect of auxin hormone; definition, Location, History and its effects on tissue culture. The third section will discuss one of the different types of cultures, callus; callus induction, culture suspension and the Importance of tissue culture. Plants produced through the technique of plant tissue culture can be stress resistant. Physical and chemical agents who have been known to cause mutations are induced to achieve the desired changes hence resulting in a variety of species. As a result, this technique has also drawbacks such as plants produced have same vulnerability to pests and environmental conditions like the traditionally produced plants because they all have the same genetic material. Plant tissue culture does not change the genetic make up the plants. 1. Plant tissue culture 1.1.Definition Plant tissue culture refers to techniques of growing plant cells, tissues, organs, seeds or other plant parts in sterile environment in an artificial nutrient medium. It may also be defined as cultural techniques for regeneration of functional plants from embryonic tissues, tissue fragments, and callus or from protoplast. 1.2. Overview Plants are made up of small components such as cells, organs or tissue that can be manipulated to grow into complete plants. Without molecular genetics and plant culture, it would be void to discuss genetic engineering as they are core to this. The technique of plant tissue culture involves obtaining a piece of plant such as an embryo and dipping it in sterile nutrient median and the science behind it. This technique allows production of extra copies of plants; enhance maturity, and production of plants without the use of seeds (Razdan, 2003). Gottlied Haberlandt report on culture of leaf mesophyll tissue and hair cells, which happed close to the end of 20th century, is considered the first successful cell and plant tissue culture. He drew the ideas from previous researches and knowledge on the biology of plants. The cells cultured by Habertlandat did not divide and hence no fruits were achieved. It was possible that plant growth regulators (PRs), which are necessary during cell division proliferation and embryo induction were absent in the culture medium. He was frustrated by this but his ideas have been used by other scientists. One of his students reported the growth of isolated root tips on a medium consisting medium salts (Robert & Dennis, 2005). In the 1930s there was progress in innovative plant tissue culture techniques after the B vitamins and natural auxin were discovered as a necessity in the growth of isolated tissues that contain meristem. Walden and Wingender (1995) reported thiamine as promoting growth on isolated tomato root tips. The first PGR, indoleacetic acid (IAA) was discovered after a series of ingenious experiments with Oat seedlings. IAA occurs naturally and is a member of auxins, which is a class of PGR. The IAA stimulated growth in excised roots. Johannes and associates obtained seedlings by enriching culture media with milk obtained from coconut, usual salts, vitamin and other nutrients. Organs were formed from cultured tissues and organs as demonstrated by Folke Skoog. Cytokinins such as adenine and kinetin were discovered to have vitro shoot-promoting effects for the rapid propagation of plants. This was so especially for the very important agronomic and horticultural cultivators. Gamborg (2002) investigated the culture of embryogenesis tissue. In order to fulfil Haberlandt’s objectives, root tips were to lead to organogenesis. Lainbch obtain hybrids after the maturity of culture medium. He isolated zygote embryos from non-viable seed of linum perenne x Laustriacum and raised zygote embryos. The process of plant tissue culture includes: nutrient medium preparation, explants isolation, equipment and explants sterilisation including sterilisation of the medium, inoculation, incubation, hardening and the establishment of the plant in the field respectively. 1.3 Plasticity and totipotency Plasticity is the ability to modify or alter, which is a very important factor in the plant tissue culture. This will be clear in plant by ability to change the circumstances of media plant to be suitable to environment, specially the regeneration and plant tissue culture. The other main factor is totipotency which is maintaining the genetic potential. Plant cells are said to be totipotent, which is the ability of undifferentiated plant tissue to differentiate into functional plants when cultured in vitro under artificial conditions (Slater et al. 2008). 1.4 Particles of the Tissue Culture Medium The tissue culture contains ninety five percent of water, sugars, vitamins, PGRs, micro and macro nutrients and organic material. A gelling agent, plant growth regulators and antibiotics are part of the culture media. The inorganic components are macro and micro salts. Their concentration is dependant on the species. A plant species is used to induce callus tissue. The success in the production of callus is dependant on the qualities of a plant and its species. The growth of callus from woody plants is slow. Explants materials from fresh and young explants are preferred. After the explants materials are collected, they are sterilised by use of sodium hypochlorite, ethanol and other chemicals with the aim of eliminating micro organisms (Misawa, 1994). Different types of salt media, which are made from inorganic compounds, have been designed for callus induction from explants, cultivation of the callus and suspended cells. Murashige and Skoog (MS) developed for tobacco tissue culture has a high nitrate, potassium (see table 1), and ammonia concentration. Another medium is B5 medium which has lower levels of inorganic nutrients than MS medium. Instead of using different types of basal media, it is preferable to use one or two types in combination of various types and concentrations of phytohormones. To obtain higher growth rate and levels of products, the medium found as most suitable is optimised (Misawa, 1994). The complex salt mixtures include mineral irons as well as essential elements needed for plant nutrition including the physiological functions of these salts. They are grouped in to iron source, macronutrients and micronutrients (see table 2) (Ramage and Williams 2002). Table 1: appears some of the elements that are essential for plant Element Function Potassium Regulates osmotic potential, principal inorganic cation Iron Electron transfer as a component of cytochromes Source: Slater et al (2008) Carbon is very necessary in the medium. It can is obtained from 2 to 4% sucrose or glucose added in the medium. For production of useful metabolites efficiently, a most efficient carbon source and optimal concentration is chosen. This depends on the products that one wants to achieve and the species of the plant. Inexpensive but efficient source have much economic benefits. Sucrose is relatively stable and easily assimilates (Ramage and Williams, 2002). Agar is a commonly used gelling agent which is produced from seaweeds. Other gelling agents include purified agar or agarose (Ramage and Williams, 2002). Thiamine (vitamin B1) is an essential vitamin for most plant cells. In some cases, vitamins stimulate growth and are hence very necessary. The vitamins in medium include: nicotinic acid, myo-inosotol (considered a B vitamin), pyridoxine and thiamine HCl. For callus tissue induction and growth promotion of cells, growth regulators known as phytohormones are required. Coconut milk has growth regulator characteristics and it is thus referred to as a growth regulators supplier (Misawa, 1994). Plant growth regulators include auxins, cytokinins, gibberllins, abscisic acid and ethylene. Cytokinins are promoters of cell division. Gibberellins act as regulators of cell elongation, thus determining the height of plants and set of fruits. Abscisic acid promotes development of distinct pathways e.g. somatic embryogenesis. It also inhibits division of cells. Ethylene controls ripening of fruits but if it builds up in tissue culture, it inhibits culture growth and development (Fowler, 2000). Antibiotics inhibit and eventually destroy other micro organism that negatively affects the growth of a plant tissue culture. This suppresses infections by bacteria, yeast and moulds in tissue culture and plant cell. After plant tissue has been transformed, they eliminate agro bacterium species (Fowler, 2000). 1.5 Preparation of Media Stock solutions made from the components described above are mixed to make the medium. 21.5 mg of kinetin is dissolved in 0.5 N HCl. Slightly heating and gradually diluting the mixture using distilled water to 100 ml achieves this. Appropriate carbon and a given volume of each stock solution are added and mixed. 0.2 NKOH or 0.2 N HCl is vital in order to regulate the PH to 5.5. Deionised or distilled water is then added to the mixture till the required volume is achieved. For solid medium, 0.6 -1.0% volumes or weight of Agar is added. This medium is distributed into vessels and then sterilised using an autoclave for 15 minutes. It is sterilised at 120 C but the conditions vary depending on the volume of the vessel and the amount of medium (Misawa, 1994). 2. Auxin Hormone 2.1Overview The word auxin originated from the Greek word: Auxein, which means to grow or enlarge. They control fundamental process like cell division and elongation, at cellular level These are hormones found in plants necessary in formation of roots and shoot. It is necessary in phototropism, fruit development, abscission, initiation and development of roots, gravitism, embryo development and providing a shade avoidance effect. The auxin gradients are necessary in guiding embryo patterning into parts that become organs from zygotes first mitotic division (Davies, 2004). On the molecular level auxin includes an aromatic ring and a carboxylic acid group (see figure 1). Auxin indole-3-acetic acid (IAA) is the most significant and major organ in the auxin family (Srivastava, 2002). 2.2Definition Auxin is a plant protein that controls the amount, type, and direction of any plant growth. The IAA in the auxin includes both natural substances, such as indole-3-acetic acid(IAA) ,4-chloro-indole-3-acetic acid(4-CI-IAA) and phenylacetic acid (PAA) (see figure2) and related synthetic compounds that have similar effects such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy acetic acid (2,4-D)( Takahashi,1986). Source: Takahashi (1986). 2.3Location Auxins are located in; meristematic tissues(see figure 3) that are in shoot(see figure 4) and root; leaves which are young and mature; mature root cells; or They can be transported throughout the plant from the apices to other parts. (Davies,2004) Source:http://www.uic.edu/classes/bios/bios100/summer2002/meristem.jpg 2.4The History of Auxin Auxins were the first plant hormones to be discovered by Charles Darwin, who worked on plant hormone research. In 1880, he first described the effects of light on the movement of canary grass (Phalaris canariensis) coleoptiles, that is special leaf originating from the first node ,which protects the new leaf (see figure 4). When the light shines, in unidirection on the coleoptile, the coleoptile curves to the direction of the light, however when the tip of the coleoptile is covered by aluminum foil, no curving occur towards the light. On cutting the tip of the coleoptile, the bending did not happen indicating that the tip of the coleoptile was responsible for the physiological reaction, of the plant bending towards the light. In 1885, Salkowski discovered indole-3-acitic acid (IAA). He found out that IAA is the major auxin involved in physiological processes in the plants. That means IAA is responsible for the physiological response of the plant bending towards the light. In 1907, Fitting made incisions on dark side of the plant but he found auxin hormone able to cross around incision and led curvature. In 1913, Boysen-Jensen used mica insert in pieces of block and put it on light side and showed the bending but when he put the block on dark side the bending did not happen. This was a modification of Fitting experiment. Later, he made another experiment, whereby he removed the tip of coleoptiles and insert gelatin between tip and plant stump causing the curvature towards the light. Source: http://botit.botany.wisc.edu/images/130/Early_development/Corn_(Zea)/Coleoptile_MC_.low.jpg The study by Paal, in 1919, shows that the coleoptiles tip cut and placed on one side of stump caused bending in absence of the unidirectional souce of the light. In 1926, research by Fritz Went suggested that drawn attention to the fact that put agar blocks under coleoptile tips for a period of time And following that he removed them to put them on stem to resume growth(see figure 5)(Srivastava,2001). Fig.(5):Important experiments of auxin in plant Source: Srivastava (2001) 2.5 Plant growth regulators and tissue culture Auxin plays a major role in plant tissue culture. Accumulation of auxins initialises new leaves formation in apical meristem. According to Gautheret findings, auxin enhances the proliferation of cambial cultures thus enabling preparation of subcultures. Media enriched with auxins makes it possible to cultivate plant tissues for a long time. It is a modulator of plant growth and development. The hormone auxin controls plant morphogenesis. However, the growth is as a result of an inter play of different hormones which must be in a balance. It is widely agreed that the percentage of auxin to cytokinin dictates the growth of roots or shoots (Pierik 1999, Slater et al. 2008) .If auxin was high to cytokinin ratios that leads to encourage root development but low ratios of auxin promote shoot development. In contrast, the growth of the callus is without differentiation when the intermediate ratios are used. (See fig.6). The auxin (IAA) is unstable to both light and heat thus its use is limited. Amino acid conjugates of IAA are occasionally used to alleviate the problem instability as they are more stable to light and heat. Auxin is also used in increasing the fruit size of the plant tissue culture. Synthetic auxins have been developed such as used in increasing fruit size in citrus flowering. However, this usually relies on several factors. These Factors include; the water level and dry matter. Auxin analogies that are promising are used in regulation of productivity in potatoes. Other auxins increase the number of tubers, activate and accelerate tuber formation (Slater et al. 2008). 3. Culture types 4. There is several plant transformation techniques employed in plant tissue culture. One of the techniques is callus induction. 3.1 Callus Induction Sterilisation of explants is done by use of 2% Sodium hypochlorite solution or with 70% solution of ethanol. Plant species determines the period after which the submerged plant is removed. Sterile water is then used to rinse it to prevent contamination. After that, sterilised plant part is cut using a sterilised scalpel approximately to length of 1cm. It is then placed on the solid medium, which is in a Petri-dish or a flask. The sterilised plant is incubated, under aseptic conditions, on the solid medium at 25° C for a number of weeks. A callus is produced after the incubation period and small pieces of the callus is transferred to fresh medium as subcultures. Subsequent transfers are done severally until the callus is soft and fragile. The containers are kept closed and can only be opened in a laminar flow hood. This is highly efficient especially in filtration. This is done to maintain sterility of media and the plant species by preventing contamination with airborne organisms. To kill fungal and bacterial contaminants, nutrient medium containers are sterilized under extreme heat ant pressure. The culture is stored where there are lights and is closely monitored. Furthermore, the humidity level is lowered gradually in preparation of transferring plant grown in laboratory condition to the soil conditions. The soils must be selected carefully with regard to the plant species (Chawla, 2002). 3.2 Culture Suspension There are two categories off callus culture; friable and compact. Densely aggregated cells are found in compact callus while loosely associated cells are found in friable callus. Suspension culture cells have higher growth rate compared to that of the solid culture. It is thus prefer on wide scale manufacture of metabolites. A section of callus is placed in a liquid medium that is already in vessel. Cultivation is generally at 100 r.p.m and at 25° C on rotary shaker but conditions depends on plant species among other factors. A fine cell suspension is established after sub culturing for several generations. The suspension contains single cells and small cell aggregates. The medium used depend on the time needed and the species of explants (Chawla, 2002). 3.3The importance of plant tissue culture Tissue culture aims at producing natural secondary metabolites with characteristics of the explants. This technology is applied in increased production of new or natural compound using cell cultures from higher plants. Transformable precursors feed in the culture medium cultures are to achieve this and the process involved is biotransformation. This skill is of importance in the medical field. Bacterial extract is sterilized and filtered and the cells are cultured in liquid medium. Genetics of cultured cells are being modified through: Selection and mutagenesis of cell lines found in cell suspension culture; Transplanting foreign genetic material found in protoplasts during of genetic engineering; and Somatic hybridization through the fusion of plants protoplasts that are distantly related with the aim of genetically diversifying of hybrids. The technology of plant tissue culture has numerous pros and cons. One advantage is its use in production of species of crop that are more drought resistant. This is a vital due to changing climatic conditions. The growth of drought resistant crops is important in dealing with the issue of food insecurity. The species produced are also more resistant to diseases, chemical pesticides, and soil conditions and herbicides thus reducing losses during crop cultivation. Foreign genes can be transplanted into protoplast. Genes of different unrelated species are brought together by hybridization through fusion of protoplast hence a hybrid is created. Biotechnology using tissue cultures techniques also aims at producing plants capable of providing own usable nitrogen. The species grown in the laboratories are important in recovery of endangered species. The growth, germination and multiplication of these species in laboratories ensure satisfactory supplies for nurseries and institutions of higher learning. This reduces cases of buying wild plants. The technology is helpful in the study of genetically modified studies. Incases where plant is infected, new plants free from infection but with similar characteristics as the infected can be produced. Growth conditions are controlled hence making synchronization of growth and development possible. This enhances acquisition of plants that have optimal characteristic. Original tissue is in small pieces and can be taken from lateral bud, root tissue, shoot tip or stem. Original plant is maintained during the process of micro propagation. Adventitious shoot proliferation and the proliferation of lateral bud bring about large increase of shoot that later produce root. Plantlets obtained or micro cuttings have responded well while being grown in the soil. There is acceleration in asexual propagation and plants respond the same way as plants that are vegetative propagated from their own roots. Plant can be grown without seasonal interruption under green house conditions. Controlled manipulations in plant lead to disarrangement, reorganization and rearrangement of plant constituents. Plant tissue culture is unique since it is a technique that is based on selection. It is thus possible to obtain, retain and maintain propagation in large quantities of plants free from pathogens. Resulting plantlets are tested for possibility of having any pathogen present in them. If the pathogens are present, the plantlets are destroyed. This is means of eliminating diseased plants from the cycle of circulation. However, much study is required on this area. Plant tissue culture also has disadvantages. Plants produced all have the same genetic material. This means their vulnerability to infections; pests and environmental conditions are similar. In case of hazardous conditions, there is massive loss of crops as all are affected. The technique can also lead to undesirable trait in the plant resulting from melolic division. Moreover, reduction in plant diversity that can be irreversible elimination of some alleles from the pool of genes. The plants might also have undesired characteristic and these have been seen to affect human health negatively. If care is not taken, there is possibility of developing poisonous plant that can easily lead to death if consumed. The plant tissue culture requires a lot of attention in all processes. Non-expertises are not mandated in the field. The machines involved incur the cost though if the desired plant culture quality and quantity are achieved, the benefits outweigh the cost of production. This technique is time consuming during the culture preparation and development period. However, growth of the plant is fast compared to the growth of plants from their original seedlings. The technique requires skilled technique and hence can only be undertaken or supervised by professional in the field of biotechnology (Sugiyama, 1999). Organic supplements such as casamino acid, yeast extracts, malt, and Coconut milk are added as growth stimulants. These components increase the growth rate of the plant tissue culture as well as that of the plant in the field (Sugiyama, 1999). Tobacco plants from single cells Appendix Table 2: media for plant tissue culture Essential element Concentration in stock solution (mg/l) Concentration in medium (mg/l) Macronutrients NH4NO3 33000 1650 KNO3 38000 1900 CaCl2.2H2O 8800 440 MgSO4.7H2O 7400 370 KH2PO4 3400 170 Micronutrients KI 166 0.83 H3BO3 1240 6.2 MnSO4.4H2O 4460 22.3 ZnSO4.7H2O 1720 8.6 Na2MoO4.2H2O 50 0.25 CuSO4.5H2O 5 0.025 CoCl2.6H2O 5 0.025 Iron sourcec FeSO4.7H2O 5560 27.8 Na2EDTA.2H2O 7460 37.3 Organic supplement Myoinositol 20000 100 Nicotinic acid 100 0.5 Pyridoxine-HCl 100 0.5 Thiamine-HCl 100 0.5 Glycine 400 2 Carbon sourced Sucrose Added as solid 30 000 Source: Slater et al(2008) Meristematic tissues: are find in all plants consisting of indistinct cells where growth zones. http://en.wikipedia.org/wiki/Meristem Plant tissue culture and food security. According to FAO, food security is ability of all human beings to get food at all times, since it is essential for their health and active life. Factors that are vital in the determination of food security of any country or region include stability of production, accessibility of food by all members of the society and the availability of the food itself. Due to changing climatic conditions and the ever diminishing land resources, which are vital in the cultivation of food crops, tradition methods of crop production cannot be relied upon. In order to meet the ever increasing demands for food crops due to increasing human population, there is need to concentrate on research that could improve the performance of these crops. Plant tissue culture should be looked at as one of the modern tools aimed at achieving these objectives. Plant tissue culture is an integral part of biotechnology, and is aimed at propagation of genotypes that of high value, production of disease free crops, productions of metabolites that are secondary etc. Plant tissue culture forms an essential step in the process of development of hybrid plants unrelated parents and genetically modified plants. It is also an important and cost-effective method which has shown a great impact in the plants that are vegetatively propagated. Tissue culture is the most effective method for production of large numbers of plants that are identical for a given market. In conjunction with genetic transformation, plant tissue culture offers an effective tool for large scale production of the transgenic crops. Production of disease free crops through plant tissue culture reduces production costs at the same time increasing yields. Tissue culture offer plant genetic engineers opportunity to introduce desirable genes to plants resulting in plants with superior characteristics like disease, chemical, saline and drought resistance. All these may result in increased food production and hence security as compared to tradition methods of plant production. References Slater, A., Scott, N. W. & Fowler, M. R. (2008), 'Plant Biotechnology' The genetic manipulation of plants, 2nd ed, Oxford University Press, The United States. Sugiyama, M. (1999), ‘Organogenesis in vitro.’ Current Opinion in Plant Biology, vol. 2, pp. 61–4. Razdan, M. K. (2003), Introduction to Plant Tissue Culture, 2nd ed, Science Publishers Inc. Walden, R. & Wingender, R.(1995), ‘Gene-transfer and plant-regeneration techniques.’ Trends in Biotechnology, vol.13, pp. 324–31. Gamborg, O. L. (2002), ‘Plant tissue culture. Biotechnology. Milestones.’ In vitro Cellular and Developmental Biology—Plant, vol. 38, pp. 84–92. Misawa‏, M. (1994), Plant Tissue Culture: an Alternative for Production of Useful Metabolites, Food & Agriculture Org. Srivastava, L. M. (2002), Plant Growth and Development :Hormones and Environment, Academic Press. Davies, P. J. (2004), Plant Hormones: Biosythesis, Signal Transduction, Action, Springer. Takahashi, N. (1986), Chemistry of Plant Hormones, CRC Press. Chawla, H. S. (2002 ), Introduction to Plant Biotechnology‎, 2nd ed, Science Publishers. Robert, N. T. & Dennis, G. J. (2005 ), Plant Development and Biotechnology, CRC Press, London. Pierik, R. L. (1999), In Vitro Culture Of Higher Plants, 4th ed, Springer, Read More